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Short-Term Clinical Exposure Evaluation of a Second-Generation Electrically Heated Cigarette Smoking System

From Philip Morris USA, Richmond, Virginia.

Address for reprints: Hans J. Roethig, Philip Morris USA, Research Center, 4102 Commerce Road, Richmond, VA 23234; e-mail: Hans-Juergen.Roethig{at}pmusa.com.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
This randomized, controlled, forced-switching, open-label, parallel-group study in 100 adult male and female smokers of conventional cigarettes evaluated 8 biomarkers of tobacco smoke exposure. After baseline exposure determinations, adult smokers were switched to a second-generation electrically heated cigarette smoking system (EHCSS) for 8 days in a clinical setting. After 8 days of smoking the EHCSS biomarkers of exposure decreased by 43% to 85% compared to baseline. After correction for residual effects (carryover effects due to long elimination half-life and non-tobacco-confounding sources of exposure), reductions in exposure ranged from 59% to 97%. Results from this short-term clinical exposure study indicate that switching from a conventional cigarette to a second-generation electrically heated cigarette smoking system substantially reduced the exposure to several measured potentially harmful constituents of tobacco smoke.

Key Words: Biomarkers • exposure evaluation • controlled smoking

Smoking cessation is the best way to reduce the risk of disease associated with tobacco smoke. However, fewer than 10% of smokers who attempt to quit each year succeed.² Because so many smokers cannot or will not quit, members of the public health community have included "harm reduction" strategies, which may include product innovations, as a "subsidiary" component of public heath policy toward tobacco.^2(p201)

In vitro investigations showed that the lower pyrolysis/combustion temperature from a first-generation electrically heated smoking system (EHCSS) resulted in significant reductions (25%-90%) in 44 measured mainstream smoke constituents relative to mainstream smoke from a University of Kentucky standard reference cigarette (1R5F).^6,7 A prior clinical exposure study of a first-generation EHCSS demonstrated 70% to 90% reduction in exposure to CO, nicotine, and mutagenic substances in adult smokers who were switched from a conventional cigarette to the EHCSS for 8 days in a controlled clinical setting.⁸

This second-generation EHCSS has further reductions in smoke constituents,^6,7 lower resistance to draw, and improved taste compared to the first-generation EHCSS.

The purpose of this study was to assess changes in biomarkers of exposure when adult smokers of conventional cigarettes were switched for 8 days in a controlled clinical setting to the second-generation EHCSS and compared to adult smokers who continued to smoke a conventional cigarette of 11 mg FTC (Federal Trade Commission) tar delivery (CC1) or who switched to a conventional cigarette of very low tar delivery (1 mg FTC tar, CC2) or to no smoking. This study design included 2 EHCSS groups. One group (EHCSS [CS]) smoked the EHCSS under controlled smoking conditions as previously described.⁸ The second group (EHCSS [UCS]) smoked the EHCSS under uncontrolled conditions, up to a maximum of 60 cigarettes per day at any time between 07:00 and 23:00, to investigate whether adult smokers in a clinical setting would increase the number of cigarettes smoked per day when switched to the EHCSS. The EHCSS is designed to deliver 8 puffs per cigarette, whereas evaluations of puffing profiles of adult smokers have indicated that they take, on average, about 11 puffs from a conventional lit-end cigarette.^9,10

The biomarkers of exposure assessed in this study included urine nicotine and 5 major metabolites, expressed as nicotine equivalents and reflecting 80% to 90% of the nicotine absorbed¹¹; plasma cotinine; 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and its glucuronides (total NNAL), metabolites of the particulate-phase smoke constituent NNK⁴; 1-hydroxypyrene (1-OHP) and its glucuronides and sulfates (total 1-OHP),⁵ metabolites of pyrene, a polycyclic aromatic hydrocarbon; carboxyhemoglobin (COHb), a biomarker for carbon monoxide; 3-hydroxypropylmercapturic acid (3-HPMA),⁴ a metabolite of acrolein; S-phenylmercapturic acid (S-PMA),⁵ a metabolite of benzene; and urine mutagenicity, reflective of compounds that are active in a modified Ames assay.^12-14

Smoking topography parameters were measured to characterize puffing behavior.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Subjects
Adult male and female volunteers 21 to 65 years of age who were in good general health and who had smoked 10 to 30 conventional cigarettes with 7 to 12 mg tar delivery (FTC) daily for at least the preceding 12 months were recruited through local advertising by the clinical study center. Each subject gave written informed consent and was paid for study participation.

To reduce variability, subjects were asked to smoke the reference cigarette (CC1) as their exclusive brand for at least the 4 weeks preceding the start of the study. Subjects indicated that they did not use any tobacco- or nicotine-containing products other than manufactured cigarettes for at least the 3 months prior to the start of the study. Exclusion criteria included clinically significant renal, liver, metabolic, cardiac, and pulmonary disease and illicit drug use. Women of childbearing potential were excluded if they were pregnant, lactating, or intended to get pregnant during the study.

Concomitant medications in stable doses to treat an investigator-approved condition (eg, hypertension, seasonal rhinitis) were generally permitted. Hormonal contraceptives and hormonal replacement therapy, occasional over-the-counter analgesics, antacids, antihistamines, and nasal decongestants were permitted. Antidiabetic and bronchodilator medications were not permitted.

Study Design and Conduct
This study used a randomized, controlled, forced-switching, open-label, parallel-group, single-center design (Figure 1). After giving informed consent and passing screening, 130 adult volunteers were admitted to the clinical center for an acclimation day followed by a baseline day and remained confined in the clinical center through day 8. After baseline investigations, 100 subjects were randomly assigned into 1 of 5 study groups: EHCSS controlled smoking (CS), EHCSS uncontrolled smoking (UCS), CC1, CC2, and no smoking. Assignment to study groups was stratified by gender and cigarette consumption on the acclimation day (10-19 cigarettes/day and 20-30 cigarettes/day). Meals served during the study were standardized to minimize dietary confounding in the biomarker assessments.

View larger version (25K): [in this window] [in a new window]   Figure 1. Study design. On day 1, CC1 smokers were randomized into 5 groups: 20 continued smoking CC1; 20 each were switched to CC2, EHCSS (CS), or EHCSS (UCS); and 20 stopped smoking. CC1 is a conventional cigarette brand, CC2 is an ultra-low-delivery conventional cigarette, and the EHCSS is the electrically heated cigarette smoking system. CS, controlled smoking conditions; UCS, uncontrolled smoking conditions.

Blood samples for carboxyhemoglobin analysis were obtained at 07:00, 11:00, 15:00, 19:00, and 23:00 at baseline and on days 1, 3, and 8. Blood samples for cotinine analysis were obtained daily at approximately 19:00 from baseline through day 8.

All urine voided by each subject was collected daily from 07:00 to 07:00 from baseline through day 8 and refrigerated at 2°C to 8°C. Urine aliquots for biomarker analysis were stored at –20°C or –70°C, as appropriate for the biomarker to be analyzed. Urine was assayed for nicotine, cotinine, trans-3'-hydroxycotinine, and their glucuronides and total 1-OHP at baseline and on days 1 through 8; total (free and conjugated) NNAL and urine mutagenicity at baseline and on day 8; and 3-HPMA and S-PMA at baseline and on days 3 and 8.

The study was approved by the local institutional review board and conducted at MDS Pharma Services (Lincoln, Neb) in accordance with good clinical practice and the Declaration of Helsinki.^15,16

Smoking Topography
Each subject smoked the first cigarette each morning and the first cigarette after lunch using the CReSSmicro device (Plowshare Ltd, Baltimore, Md) to obtain smoking topography data (number of puffs per cigarette, puff volume, puff duration, interpuff interval).

Test Products
Tar, nicotine, and CO deliveries in mainstream smoke for each of the 3 cigarette products used in this study were determined from smoking machines operated under FTC conditions as follows:

• CC1 (Marlboro Lights): 11 mg tar, 0.8 mg nicotine, and 12 mg CO
  
• CC2 (MERIT Ultima): 1 mg tar, 0.1 mg nicotine, and 4 mg CO
  
• EHCSS (ACCORD version JLI): 3 mg tar, 0.2 mg nicotine, and 0.7 mg CO
  

Bioanalytical Methodology
All biomarkers were measured by liquid chromatography/tandem mass spectrometry (LC/MS/MS) methods (except for mutagenicity and COHb), validated according to the Food and Drug Administration (FDA) guidance.¹⁷ Sample matrix, method, lower limit of quantification (LLOQ), and method precision for each biomarker are shown in Table I.

COHb and urine mutagenicity were measured as described previously.⁸

Urine Nicotine and 5 Metabolites
Nicotine, nicotine-N-glucuronide, cotinine, and trans-3'-hydroxycotinine were analyzed by LC/MS/MS, and total cotinine and total trans-3'-hydroxycotinine were measured in a separate LC/MS/MS analysis after enzymatic deconjugation. After the addition of internal standards (nicotine-d₃ (methyl-d₃), cotinine-d₃ (methyl-d₃), trans-3'-hydroxycotinine-d₃ (methyl-d₃), and nicotine-N-glucuronide-d₃ (methyl-d₃)) to a 1.0-mL volume of urine and basification to pH 10.0, the sample was loaded onto a preconditioned Oasis HLB solid-phase extraction (SPE) cartridge. The sample was washed with NH₄OAc (pH 6.6) and then eluted with methanol. Extracts were evaporated to dryness and reconstituted in 200 µL of methanol. A 2-µL aliquot of the methanol solution was injected onto a 4 x 2-mm Phenomenex silica precolumn attached to a 50 x 3-mm, 5-µm Varian MetaChem Inertsil Silica analytical column. A mobile phase consisting of 90% acetonitrile (ACN)/10% water/0.05% trifluoroacetic acid eluted the analytes into a PE Sciex API 3000, using an ESI interface. Positive ions were monitored in the multiple-reaction monitoring (MRM) mode:

• Nicotine and nicotine-d₃: m/z 163.1 132.2 and 166.1 132.2
  
• Nicotine-N-glucuronide and nicotine-N-glucuronide-d₃: m/z 339.2 163.1 and 342.2 166.1
  
• Cotinine and cotinine-d₃: m/z 177.0 80.1 and 180.0 80.1
  
• Trans-3'-hydroxycotinine and trans-3'-hydroxycotinine-d₃: m/z 193.2 80.1 and 196.1 80.0
  

For the analysis of total cotinine and total trans-3'-hydroxycotinine, a 0.2-mL aliquot of urine was incubated at 37°C for 20 to 24 hours with ß-glucuronidase (type H-1, Helix pomatia [Sigma, St. Louis, Mo]). After adding internal standards, the resulting solution was analyzed for total cotinine and total trans-3'-hydroxycotinine, as described above. The 24-hour excretion of nicotine, nicotine-N-glucuronide, total cotinine, and total trans-3'-hydroxycotinine was calculated from the measured concentration and 24-hour urine volume, converted into moles excreted in 24 hours, summed to yield moles of nicotine equivalents (NE), and multiplied by the molecular weight of nicotine to yield NE in milligrams.

Plasma Cotinine
Heparin plasma samples were aliquoted into a 96-well sample plate. A protein precipitation extraction and filter procedure was used for sample purification. Samples were injected onto a Varian MetaChem Inertsil Silica 50 x 3-mm, 5-µm analytical column and eluted with ACN/water/trifluoroacetic acid into a PE Sciex API 4000 using an ESI interface. Positive ions were monitored in the MRM mode, and the following transitions were recorded: cotinine and cotinine-d₃: m/z 177.1 80.1 and 180.1 80.1.

Urine Total 4-Methylnitrosamino-1-3-(pyridyl)-1-butanol (NNAL)
After the addition of the internal standard (d₃-NNAL), a 2-mL urine sample was pH adjusted and incubated with ß-glucuronidase (type H-1, H. pomatia) to cleave the glucuronide from the NNAL-glucuronide conjugate. The hydrolyzed sample was extracted using mixed-mode solid-phase extraction. The extract was injected onto a Keystone Hypersil silica column with a guard column and eluted with ACN/H₂O/formic acid (70:30:0.7, v/v/v) mobile phase into an AB/MDS Sciex API 4000, using an ESI interface. Positive ions were monitored in the MRM mode for the reactions of NNAL and d₃-NNAL: m/z 210.1 180.1 and 213.1 183.1.

Urine Total 1-Hydroxypyrene (1-OHP)
After the addition of the internal standard (d₉-1-OHP), the sample was pH adjusted and incubated with ß-glucuronidase (type H-1, H. pomatia, which contained both ß-glucuronidase and sulfatase) to cleave the 1-OHP from its glucuronide and sulfate conjugates. The hydrolyzed sample was extracted using C₈ solid-phase extraction columns. The extract was injected onto a Thermo Hypersil-Keystone Kromasil C₁₈ column and eluted with ACN/10 mM ammonium acetate, pH 5 (75:25, v/v), mobile phase into a PE Sciex API 4000, using an ESI interface. Negative ions were monitored in the MRM mode: 1-OHP and d₉-1-OHP: m/z 216.8 188.9 and 226.0 198.0.

Urine 3-Hydroxypropylmercapturic Acid (3-HPMA)
After the addition of the internal standard (d₆-3-HPMA), the sample was acidified before separation on an Oasis HLB mixed-mode solid-phase extraction cartridge. The samples were eluted with 0.5% ammonium hydroxide in acetonitrile before evaporation and then reconstituted with 80:20 methanol/water. The sample was injected onto a Thermo Hypersil BioBasic AX column (50 x 4.6 mm, 5 µ) with a guard column and eluted with 80:20 acetonitrile/50-mM ammonium acetate (pH 4.5) mobile phase into a PE Sciex API 4000, using an ESI interface. Negative ions for the following transitions were monitored in the MRM mode: 3-HPMA and d₆-3-HPMA: m/z 220.0 90.9 and 225.8 96.7.

Urine S-Phenylmercapturic Acid (S-PMA)
After the addition of the internal standard (d₅-S-PMA), the sample was acidified before separation onto a polymeric solid-phase extraction cartridge. The sample was eluted with an ammoniated polar organic solvent before evaporation. The reconstituted sample was injected onto a Thermo Hypersil BioBasic AX column and eluted with an ACN/50-mM ammonium acetate, pH 3.5 (90:10, v/v), mobile phase into a PE Sciex API 4000, using an ESI interface. Negative ions were monitored in the MRM mode: S-PMA and d₅-S-PMA: m/z 238.1 108.8 and 243.1 114.0.

Data Analysis
Descriptive statistics were provided for the daily amount of biomarker excreted, the daily amount of biomarker excreted adjusted by the number of cigarettes smoked, and percent change from baseline in daily biomarker excretion.

Carboxyhemoglobin was measured every 4 hours from 07:00 to 23:00. The area under the curve (AUC) of the concentration versus time profiles was assessed using numerical integration (simple trapezoidal rule), and COHb AUC_(7-23) was used as the analysis variable.

A linear mixed model for repeated-measure analysis of variance was applied to percent change from baseline in daily biomarker excretion. The model included terms for study group (5 groups), study day (8 days), gender, cigarette consumption (2 levels), smoking inhalation depth (2 levels), and subject. Pairwise comparisons of the least squares means for the study groups were performed for each biomarker with a statistically significant study group effect.

The percent change from baseline to day 8 in the mean 24-hour urine biomarker excretion in each of the groups, adjusted for the residual effect in the no-smoking group at day 8, was calculated as follows:

where, for each biomarker, the baseline adjusted group mean value was calculated as

and the day 8 adjusted group mean value was calculated as

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Subjects
One hundred adult smokers (50 men and 50 women) were randomized (Table II), and 97 adult smokers (47 men and 50 women) completed the study. Two adult smokers (1 each from the CC1 and EHCSS [UCS] groups) left the study for personal reasons. One subject from the EHCSS (CS) group withdrew from the study because of arm pain and abnormal sensations after a blood draw.

Daily Cigarette Consumption
Daily cigarette consumption data are presented in Table III and Figure 2. In the EHCSS (CS), CC1, and CC2 groups, mean daily cigarette consumption remained stable from baseline through day 8, consistent with the controlled smoking conditions. Compared to baseline when subjects smoked CC1, average daily cigarette consumption increased by 51% in the EHCSS (UCS) group at day 8.

View larger version (15K): [in this window] [in a new window]   Figure 2. Daily cigarette consumption (mean ± SEM) for all groups. At baseline, all subjects smoked CC1. From days 1 to 8, all smokers except those in the EHCSS (UCS) group were limited to the maximum number of cigarettes smoked on the acclimation day (controlled smoking). EHCSS, electrically heated cigarette smoking system; UCS, uncontrolled smoking.

Nicotine Equivalents
Figure 3 shows the 24-hour urine nicotine equivalents excretion. The groups did not differ significantly at baseline when all subjects smoked CC1. From baseline to day 8, the mean 24-hour urine nicotine equivalents excretion decreased by 46% in the EHCSS (CS) group, 43% in the EHCSS (UCS) group, and 37% in the CC2 group, and it was relatively stable in the CC1 group. Compared to CC1, all changes were statistically significant (P < .0001). The difference between the EHCSS groups (CS vs UCS) in change from baseline to day 8 was not significant (P = .79), despite smokers in the EHCSS (UCS) group smoking, on average, about 50% more cigarettes daily. The apparent t_1/2 for urine excretion of nicotine equivalents, based on the amount excreted versus time curve in Figure 3 for the no-smoking group, was about 24 hours.

View larger version (15K): [in this window] [in a new window]   Figure 3. Twenty-four-hour nicotine equivalents excretion (mg/24 h, mean ± SEM). At baseline, all subjects smoked CC1. EHCSS, electrically heated cigarette smoking system.

The 24-hour urine nicotine equivalents excretion, adjusted by the number of cigarettes smoked during the 24-hour urine collection intervals, is summarized in Table IV. The mean levels of nicotine equivalents per cigarette in all groups were not significantly different at baseline when all subjects smoked CC1 (approximately 1.0 mg/cig). At day 8, the mean nicotine equivalents per cigarette decreased to 0.55 mg/cig in the EHCSS (CS), 0.36 mg/cig in the EHCSS (UCS), and 0.58 mg/cig in the CC2 groups.

Plasma Cotinine
Figure 4 illustrates plasma cotinine concentrations. There were no significant differences between the 5 groups at baseline. At day 8 (Table V), mean plasma cotinine had decreased from baseline by 53% in the EHCSS (CS) group, 50% in the EHCSS (UCS) group, and 41% in the CC2 group, and it was relatively stable in the CC1 group. Changes in plasma cotinine levels were significant in all smoking groups compared to the CC1 group (P < .0001).

View larger version (13K): [in this window] [in a new window]   Figure 4. Plasma cotinine concentrations (mg/mL, mean ± SEM). At baseline, all subjects smoked CC1. EHCSS, electrically heated cigarette smoking system.

Total NNAL (NNK Metabolite)
At baseline, there were no significant differences in total NNAL among the 5 groups. From baseline to day 8, mean 24-hour urine total NNAL excretion decreased by 52% in the EHCSS (CS) group, 60% in the EHCSS (UCS) group, 14% in the CC2 group, and 73% in the no-smoking group. It increased by 26% in the CC1 group from baseline to day 8 (Table V). The changes in total NNAL were statistically significant (P < .0001) for all groups compared to the CC1 group.

Total 1-Hydroxypyrene (1-OHP)
Figure 5 presents the 24-hour urine excretion of total 1-OHP. Total 1-OHP levels were similar in all groups at baseline while smoking CC1. At day 8, mean 24-hour total 1-OHP decreased from baseline by 66% in the EHCSS (CS) group, 72% in the EHCSS (UCS) group, 44% in the CC2 group, and 71% in the no-smoking group (Table V).

View larger version (14K): [in this window] [in a new window]   Figure 5. Urine total 1-hydroxypyrene (1-OHP) (ng/24 h, mean ± SEM). At baseline, all subjects smoked CC1. EHCSS, electrically heated cigarette smoking system.

Urine Mutagenicity
At baseline while smoking CC1, median urine mutagenicity was comparable in all groups, and it remained stable between baseline and day 8 in the CC1 group. From baseline to day 8, mean urine mutagenicity decreased in all other groups: by 67% in the EHCSS (CS) group, 68% in the EHCSS (UCS) group, 39% in the CC2 group, and 75% in the no-smoking group (Table V).

Carboxyhemoglobin
The COHb AUC_(7-23) results are shown in Figure 6. At baseline, there were no significant differences between the 5 groups in mean COHb AUC_(7-23). At day 8, mean COHb AUC_(7-23) decreased from baseline by 86% to 87% in the EHCSS groups, 27% in the CC2 group, and 92% in the no-smoking group, and it remained stable in the CC1 group (Table V). The changes in mean COHb AUC_(7-23) were statistically significant in all groups compared to CC1.

View larger version (12K): [in this window] [in a new window]   Figure 6. Mean carboxyhemoglobin (COHb) concentration (%) at baseline (left) and day 8 (right). At baseline, all subjects smoked CC1. EHCSS, electrically heated cigarette smoking system.

3-Hydroxypropylmercapturic Acid (3-HPMA)
The 24-hour urine excretion of 3-HPMA is shown in Figure 7. At baseline, 3-HPMA levels were similar among all study groups and remained stable over the 8 days in the CC1 group. From baseline to day 8, mean 24-hour urine 3-HPMA excretion decreased by 57% in the EHCSS (CS), 48% in the EHCSS (UCS), 25% in the CC2, and 81% in the no-smoking groups (Table V); all changes were significant compared with CC1 (P .0001). The decreases in the 2 EHCSS groups were not significantly different.

View larger version (12K): [in this window] [in a new window]   Figure 7. Urine 3-hydroxypropylmercapturic acid (3-HPMA; µg/24 h, mean ± SEM). At baseline, all subjects smoked CC1. EHCSS, electrically heated cigarette smoking system.

View larger version (10K): [in this window] [in a new window]   Figure 8. Urine S-phenylmercapturic acid (S-PMA; µg/24 h, mean ± SEM). At baseline, all subjects smoked CC1. EHCSS, electrically heated cigarette smoking system.

Safety Evaluations
No significant group-related differences were observed for vital signs, electrocardiogram (EKG), hematology, clinical chemistry, and urinalysis investigations. Forty-six subjects (46%) experienced at least 1 adverse event during the trial, with headache the most commonly reported adverse event across all study groups. All adverse events were mild or moderate in severity, and none was considered likely to be study product related by the investigator.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
This study of adult smokers was conducted to evaluate, in a controlled clinical setting, biomarkers of exposure reflecting both the particulate and gas phases of cigarette smoke after switching to an electrically heated cigarette smoking system compared to continuing to smoke a conventional cigarette. This study design has been previously demonstrated to evaluate exposure to different types of cigarettes.⁸ This study design included a forced switch from a conventional cigarette (CC1), smoked by all subjects for at least the preceding 4 weeks, to an electrically heated cigarette smoking system, to a very low-tar delivery (FTC) conventional cigarette (CC2), or to no smoking.

"Controlled smoking" conditions were included in the design to keep the daily number of cigarettes smoked rather constant over the study, which was reflected in stable biomarker levels in the "no-change" reference group that continued smoking CC1. This allowed an evaluation of the effect of the cigarette design itself and associated smoking behavior, excluding possible compensation by number of cigarettes smoked, on biomarkers of exposure. An "uncontrolled smoking" group was included in this clinical setting to assess the effect of possible compensatory smoking behavior, of changing the number of cigarettes smoked daily. Because the EHCSS is designed to deliver 8 puffs per cigarette, whereas adult smokers in this study took, on average, about 12 puffs from a conventional lit-end cigarette, it was hypothesized that adult smokers might increase the daily number of cigarettes smoked by about 50% when switched to the EHCSS. The data show that the mean daily numbers of cigarettes smoked at baseline and on day 8 were comparable in the controlled smoking groups but increased in the EHCSS uncontrolled smoking group by about 50% on day 8. There was no uncontrolled smoking group for conventional cigarettes in this study due to study size limitations. Therefore, no final conclusion is possible regarding whether the increase in numbers of cigarettes in the EHCSS (UCS) group was related to switching to the EHCSS or a matter of the study procedures. Despite this increase in number of cigarettes smoked, the change from baseline to day 8 in measured levels of biomarkers of exposure was not significantly different between the 2 EHCSS groups. At first glance, this result is surprising; it is, however, a consistent finding, with the same effect noted for all the biomarkers included in this study. A possible explanation for this similarity in exposure despite the difference in cigarette consumption is that adult EHCSS smokers in the uncontrolled smoking group smoked differently (eg, less intensely) than did adult EHCSS smokers in the controlled smoking group. In fact, measured puff volumes, on average, were lower for adult EHCSS smokers in the uncontrolled smoking group compared to adult EHCSS smokers in the controlled smoking group (smoking topography, Table VI).

Although the biomarker levels in the CC1 group varied little over the course of the 8-day exposure period, all biomarkers of exposure to smoke constituents of both the particulate and gas phases were significantly reduced in both EHCSS groups, by 43% to 87% relative to baseline. This is consistent with a reduction in exposure for the adult smokers who were switched to the EHCSS. Even when adult smokers of the EHCSS were allowed to smoke as many cigarettes as they wished (uncontrolled smoking conditions), the reduction in exposure was substantial and similar in magnitude to that during controlled smoking conditions. It is noteworthy that the adult smokers puffed the EHCSS substantially different than their conventional cigarette, beginning with the first cigarette smoked, by taking longer puffs with more than 50% higher puff volumes. When adult smokers were switched to the EHCSS for 8 days, the reduction in levels of several biomarkers of exposure (ie, COHb AUC_(7-23) and urine S-PMA, total 1-OHP, and mutagenicity) was similar to reductions in the group that stopped smoking for 8 days, the "maximum change" reference group. Switching from an 11-mg tar delivery (FTC) cigarette (CC1) to a very low 1-mg tar delivery (FTC) cigarette (CC2) also resulted in significant reductions in biomarkers of exposure to smoke constituents, although the magnitude was lower than the reductions seen for the EHCSS. Smoking topography data showed that adult smokers changed their puffing behavior by taking longer puffs with about 20% higher puff volumes and shorter interpuff intervals, when switched from CC1 to CC2.

As reported in the Results section, total 1-OHP levels were increased in all study groups on day 5. There are several confounding sources of PAHs in the environment, the most common being grilled or broiled meats, automobile or diesel exhaust, and home heating systems. The design of this study minimized the likelihood of exhaust or incomplete combustion from heating systems being the source of the pyrene exposure. Although the meals served during the study included menu items selected to eliminate dietary sources of confounding, similar uniform increases at day 5 were seen in other studies. Changes were made in the preparation of the food served to eliminate any protein being exposed to direct heat and any cured meats, and the day 5 1-OHP exposure increase was not observed in subsequent studies.

The biomarkers of exposure measured in this study reflect the effect of switching to another tobacco product. However, there may be a carryover effect from smoking the previous conventional cigarette product, particularly if the elimination half-life of the biomarker is longer than one fifth of the 8-day exposure period, as is the case for NNAL.¹⁸ In addition, exposure to certain constituents, such as benzene, polycyclic aromatic hydrocarbons, acrolein, and CO, is not entirely tobacco smoke specific, and these confounding sources of exposure will not be influenced by switching to another tobacco product. To more clearly estimate a smoking group's exposure due solely to switching to another tobacco product, biomarkers were adjusted for the residual effect due to such carryover and confounding influences, which was estimated in the no-smoking group at day 8. Adjusting for these residual levels of biomarkers provides a more accurate measure of smoking-related exposure. The percent change from baseline to day 8 in mean 24-hour biomarkers, adjusted for the residual effect in the no-smoking group at day 8, is presented in Table V for each study group. At day 8, after adjusting for the residual effect in the no-smoking group at day 8, the EHCSS (CS) group had about 46% to 68% lower mean urine nicotine equivalents and 3-HPMA and about 66% to 95% lower mean evening COHb, urine total NNAL, S-PMA, total 1-OHP, and mutagenicity compared to baseline levels while smoking CC1. The relatively short elimination half-lives of nicotine and its metabolites and the absence of any confounding sources for nicotine in a confinement study resulted in 100% reduction in nicotine equivalents and plasma cotinine on day 8, and therefore there was no residual adjustment for nicotine or its metabolites.

Most investigations of CO exposure based on COHb include single time point measurements. Similarly to a previous research study, conducted in a different research program,¹⁹ this study incorporated 5 single time point COHb measurements over each 16-hour daily smoking interval, which permitted estimation of the integrated exposure (AUC) over the entire daily smoking interval. At baseline, while all subjects smoked CC1, a strong correlation between the results from the single time point measurements and the AUC values was observed for all time points, with the strongest relationship obtained for the 15:00 and 19:00 time points (r = 0.95 and 0.96, respectively). These results indicate that a COHb measurement from a single blood draw at 15:00 or 19:00 provides an assessment of CO exposure comparable to that determined by a 16-hour AUC measurement based on 5 blood draws throughout the smoking day.

In conclusion, all measured biomarkers of exposure were significantly reduced in adult smokers of conventional cigarettes relative to baseline after switching to EHCSS for 8 days in a controlled, confined, clinical setting. The greatest reductions, approaching 90%, occurred for COHb and S-PMA. The smallest reductions from baseline among the 8 biomarkers measured occurred for nicotine, which was reduced by 43% in EHCSS (UCS) and 46% in EHCSS (CS). Results from this clinical study confirm that lowering the temperature during tobacco combustion results in a substantial reduction in exposure, as measured by 8 biomarkers of exposure for smoke constituents of both the particulate and gas phases.

Clinical studies of longer duration in a normal life setting are necessary to evaluate exposure reduction in longer term users of the EHCSS and whether reduced exposure is associated with reduced risk of smoking-related diseases.

	ACKNOWLEDGEMENTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
The authors thank MDS Pharma Services for clinical conduct and bioanalytical and statistical analyses, as well as PK Noonan & Associates, LLC for assistance in developing this manuscript.

Financial disclosure: Financial support provided by Philip Morris USA.

DOI: 10.1177/0091270006297686

	REFERENCES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 

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